Acrosin (E.N.3.4.21.10.) is a unique trypsin-like proteinase present in mammalian sperm acrosomes. Evidence strongly suggests this enzyme is used by sperm to digest a path through the zona pellucida of the ovum, a prerequisite to fertilization. The applicant has previously purified boar acrosin and has determined that acrosin has an unusually high arginine substrate specificity. This project is designed to continue the study of homogeneous acrosin. Particular emphasis will be given to determining which active site amino acids are involved in acrosin's mechanism of action. In attempting to understand acrosin's catalytic properties comparisons to pancreatic trypsin will be made. The unique properties such as its unusually high arginine specificity and competitive inhibition by various arginine analogs will be studied. A kinetic evaluation of these properties may define agents which bind specifically at the active site of acrosin and not that of trypsin. These inhibitors will be studied to determine the site of alkylation and the specificity of inhibition. Finally, the effects of the inhibitors on the potential zona penetrating ability of spermatozoa will be evaluated by in vitro tests.